Project Abstract The Pol ?-primase synthesizes a 7-10 nucleotide RNA primer, and Pol ?-primase then extends this primer by an additional 10?20 dNMP before dissociating. Pol ?-primase is comprised of four subunits, including the Pri1 and Pri2 primase subunits, the catalytic Pol1 subunit, and the essential Pol12 regulatory subunit. The budding yeast replicative helicase, comprised of Cdc45, Mcm2-7, and GINS (CMG helicase), unwinds DNA at a replication fork. Our preliminary data demonstrate essential, novel interactions between the Pol12 subunit of Pol ?-primase and Mcm2-7 or single-stranded DNA. Furthermore, the interaction between Pol12 and Mcm2-7 is strengthened by the S phase cyclin-dependent kinase (S-CDK). Mcm proteins function as tumor markers, Pol ?-primase generates DNA mutations in the genome, and S-CDK is currently a target for small molecule inhibitors to treat cancer. Thus, a mechanistic understanding of how the Pol12 subunit of the Pol ?-primase functions in coordination with the helicase, cell-cycle kinase, and DNA may lead to improvements in how cancer is diagnosed or prevented. We will first determine how Pol ?-primase is recruited to the CMG helicase during replication initiation. I reconstituted the DNA replication initiation assay in my laboratory using purified budding yeast proteins, using reagents produced in my laboratory. I found that Pol12 binds to Mcm2-7, and I have identified point mutations of Pol12 that specifically disrupt the interaction with Mcm2-7. Expression of this mutant is lethal to yeast cells, and, we will test the hypothesis that Pol12-Mcm2-7 interaction is required for Pol ?-primase recruitment to CMG helicase during replication initiation using the in vitro and in vivo methodologies. We will also determine how Pol ?-primase is activated to synthesize RNA during replication initiation. I found that Pol12 binds to ssDNA in vitro, and I also determined that Pol12-ssDNA interaction is required for yeast cell viability. We will test the hypothesis that Pol12 interaction with ssDNA is required for Pol ?-primase RNA synthesis activity in vitro, using the replication initiation system, and in vivo, using a conditional degron strain. Determining how Pol ?-primase is recruited to the CMG, how Pol ?-primase RNA synthesis is activated, and the essential function for the Pol12 subunit will reveal a mechanistic understanding of how the CMG helicase couples to the Pol ? polymerase to initiate DNA synthesis in eukaryotes. This project will advance our understanding of how replication is initiated in eukaryotic cells, and may ultimately lead to further advances in the prognosis and treatment of cancer. This proposal will also provide excellent training opportunities for graduate and undergraduate students. !